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Detection and quantification of Rhizoctonia solani in soil by monoclonal antibody-based immuno-magnetic bead assay
PIIt
Soil Biol. B&hem. Vol. 28, No. 415, pp. 521-532, 19% Copyright 0 1996Ekvicr ScienceLtd Printedin Great Britain. AU rights reserved soo38o717(95)08164-6 0038-0717/% s15.00 + 0.00
DETECTION AND QUANTIFICATION OF RHIZOCTONIA SOLANI IN SOIL BY MONOCLONAL ANTIBODY-BASED IMMUNO-MAGNETIC BEAD ASSAY C. R. THORNTON Department Plant Sciences, University of Cambridge, Downing St., Cambridge CB2 3EA, U.K. (Accepted 20 November 1995)
Sammy-Murine monoclonal antibodies (MAbs) of the immunoglobulin classes IgM and IgA, from two different hybridoma cell lines, and rabbit (polyclonal) antisermn raised against mycehal antigens from an AG4 isolate of R. sofoni were used to develop magnetic microsphere-enzyme immunoassays (MM-EIAs) for the detection and quantitication of the pathogen in soil. The assay can also be used to differentiate R. solani anastomosis group (AG) 2-2 isolates from other AG isolates grown
in vitro. My&ii
antigens were solubilised in saline buffer containing detergent and following high speed centrifugation soluble antigen was delineated by the addition either of a mixture of specificmurine MAbs
of diffemnt immunoglobulin classes or a mixture of murine MA\, supematant and rabbit antiserum. Due to the low concentration of reactants a soluble immune complex was formed. The soluble complex was isolated by the addition of magnetic beads coated with antibodies that specitically recognised the murine IgM MAbs. The bound antibody-antigen complex was then detected using a commercial antibody-enzyme conjugate and chromogen. Copyright 0 1996 Elsevier Science Ltd
INTRODUCTION
Thornton et al. (1993) reported the development of monoclonal antibodies (MAbs) and MAb-based immunological assays for the detection of Rhizoctonia solani in soil. These assays involve the immobilisation of mycelial antigens onto solid supports (PVC micro-titre wells or PVDF membrane) using an overnight incubation step and subsequent visualisation of bound antigen by enzyme-linked immunosorbent assay (ELISA). My aim is to communicate the development of a novel immunomagnetic bead assay for the detection and quantification of R. sofani mycelial antigens in soil. Magnetic beads have been employed successfully in the medical field but have, to date, been used exclusively as a means of selective cell separation (e.g. Nilsson et al., 1987; Wynick and Bloom, 1990). Wipat et al. (1994) reported the use of MAb-coated beads in the immunomagnetic capture and concentration of spores of the bacterium Streptomyces liuidans in soil while Pain et al. (1994) used immunomagnetic separation to isolate viable intracellular hyphae of Colletotrichum linakmuthianum from infected bean leaves using MAbs. The incorporation of magnet&d beads into magnetic microsphere-enzyme immunoassay (MM-EIA) formats has been associated most notably with the detection and quantification of plant viruses (e.g. Banttari et al., 1991). To my knowledge, no studies
have reported the detection of fungal antigens in soil using an immune-magnetic bead assay. One of the most significant rate determining steps in the development of sensitive immunoassays for the detection of soil-borne fungi is the extraction of their antigens. Most assays require lengthy extraction and concentration procedures often involving nutrient enrichment, sieving, flotation, centrifugation and filtration. The immunoassays themselves often require lengthy processing times. Since most enzyme immunoassays use micro-titre plates or membranes as the immunosorbent surface, they sulfer from slow binding kinetics and small surface areas. In contrast, enzyme immunoassays that incorporate magnetic particles (l-10 pm) have larger binding surfaces and faster binding kinetics. Antibody-coated magnetic beads have the added advantage of utilising a separation step that is uniquely suited to efficiently extracting antigens from samples (l-2 ml) that contain particulate debris. Thus, it is possible to develop immunoassays that combine the extraction, concentration and detection of antigens into a single relatively simple and rapid step. A magnetic field can then be applied that pulls the beads and bound antigen to the side of a micro-centrifuge tube and the remaining soluble and particulate debris can then be decanted or aspirated. The bound antibody-antigen complex is subsequently quantified by the addition of an antibody-enzyme conjugate and chromogen using 521
528
C. R. Thornton
procedures similar to those employed double-antibody-sandwich ELISAs.
by other
METHODS
Fungal culture
Isolates of Rhizoctonia solani anastomosis groups (Table 1) were maintained on potato dextrose agar (PDA, Oxoid CM139) either in Petri-dishes or on slants at 23°C. Development of monoclonal antibodies
The R. solani-specific murine monoclonal antibodies (MAbs) and rabbit (polyclonal) antiserum, used were developed by Thornton et al. (1993); murine hybridoma cell lines EEl and EH2 that produce immunoglobulins of the classes IgM and IgA respectively, and rabbit antiserum, were raised against mycelial antigens from an R. solani AG4 isolate R3. Detection and quanttjkation of R. solani antigens in artificially infested soil by MAb-based immuno-magnetic bead assay Preparation of arttficially infested chopped potato soil. Three-hundred g sieved (0.5-l mm) and
air-dried loam soil (Cambridge University Field Station), and 75 g finely chopped potato (wet weight) were mixed thoroughly with 45 ml reverse osmosis water (R.0.H20) and autoclaved in 250 ml Erlenmeyer flasks for two consecutive 1 h periods at 121°C. Flasks were inoculated with six 3 cm mycelial plugs taken from the leading edge of 5-d-old PDA Petri-dish cultures of either R. solani (CPS( + )) or were inoculated with a similar number of uncolonised plugs (CPS( - )). The flasks were kept in the dark, at 23°C for 2 weeks. Extraction of antigen and preparation of standard curve. Five replicate 5 g fresh samples from
artificially infested and control flask cultures were placed in separate 20 ml polyvials containing 5 ml of PBST buffer (PBS, NaCl, 0.8%; KCl, 0.02%; Na2HP04, 0.115%; KH2P04, 0.02%; PBST, PBS + 0.05% (v/v) Tween-20 [polyoxyethylene(20)sorbitan monolaurate]) adjusted to Table I. Details of fungal cultures Organism Rhizockmia camtoe Rhizockmia cerealis Rhizoctonia solani
AG
I 2-l 2-2 3 4 4 5 7
R
tuliparum
Isolate
numtw
168256 RI 303 154/R20 R5 303 156/R2 1 303 138/R22 303162/R23 R3 303 159/R24 303161/R25 236841
RB, R. Baker; FMD, F. M. Dewey; IMI, International Institute, Egham, Surrey.
Source IMI FMD IMI RB IMI IMI IMI RB IMI IMI IMI Mycological
pH 8.3 with 1 M NaOH and the vials incubated for 16 h, at 23”C, with mixing. The contents of the vials were centrifuged, at 12,000 g for 20 min, and 875 ~1 of extract transferred to 1.5 ml micro-centrifuge tubes for immediate assay by magnetic microsphereenzyme immunoassay (MM-EIA). A standard curve, relating biomass of mycelium (pg dry mycelium) to absorbance value, was constructed to determine the sensitivity of the enzyme immunoassay. Sterile nitro-cellulose millipore filter papers (dia 0.44 cm, pore size 0.45 pm), overlaying PDA in 9 cm Petri-dish plates, were inoculated centrally with single 3 cm dia plugs taken from the leading edge of 5-d-old PDA cultures of R. solani. After approximately 5 days at 23°C the filters were extracted with forceps and discs of mycelium punched from the filters using a 501 micro-pipette (Camlab, Cambridge, U.K.). The mycelial discs were separated from the filters and were placed in 1 ml volumes of PBST (pH 8.3) contained in micro-centrifuge tubes and homogenised for approximately 5 min using a hand-held Eppendorf homogeniser. Cellular debris was removed by centrifugation at 12,000 g for 20 min and the supematant, containing soluble antigen, transferred to clean micro-centrifuge tubes for immediate assay by MM-EIA (format [A]) described below. Recognition of anastomosis group isolates. Differentiation of AG isolates of R. solani and related Rhizoctonia spp., by immuno-magnetic bead assay, was tested using cell-free PBST (pH 8.3) surface washings of antigens from slant cultures, diluted IO-fold into PBST, according to the MM-EIA format [A] described below. 1Uh4-EIA formats. Aliquots (25 ~1) of magnetic beads (Dynabeads M-450 rat anti-mouse IgM [Dynal (UK)] Ltd., No. 110.15) coated with rat monoclonal antibodies that specifically recognise the p heavy chain of mouse IgM immunoglobulins (approximately 10’ beads ml - ’storage buffer (0.1 M NaP04 buffer, pH7.4, containing 0.1% (w/v) human serum albumin and 0.02% (v/v) NaNj) were placed in 1.5 ml micro-centrifuge tubes. The beads were washed four times, by vortexing, with 1 ml volumes of TBS buffer (NaCl, 0.8%; KCI, 0.02%; Tris, 0.3%; pH 8.3). After each washing step the beads were seperated using a magnetic particle concentrator (Dynal, MPC-M) and the washing supematant removed by aspiration. Washed beads were finally resuspended, with vortexing, in TBST buffer (TBS + 0.05% (v/v) Tween-20, pH 8.3) with a volume equivalent to the starting volume of storage buffer. Two MM-EIA formats were tested (Fig. 1); the first, designated format [A] used a mixture of murine IgM and IgA MAbs while the second (format [B]) used a mixture of murine IgM MAbs and rabbit antiserum as follows: Step (1). To each of the tubes containing 875 ~1 of antigen extract were added either 50 ~1 each of EEl (IgM) and EH2 (IgA) MAb supematants (MM-EIA
Detection and quantiiication of Rhizoctonia solani in soil
FORMAT
529
FORMAT
[A]
colour forktion Fig. 1. Diagramatic representation
format [A]) or 99 ~1 of EEl MAb supernatant and 1 ~1 of rabbit antiserum (MM-EIA format [B]). Solutions were mixed thoroughly, by vortexing, and incubated with gentle mixing for 30 min at 23°C. Step (2). Washed beads (25 ~1) were added to each tube and after thorough vortexing the mixtures were kept for 30 min at 23°C with gentle agitation. After magnetic separation, the beads were washed free of unbound protein (1 ml each wash) by two cycles of separation and re-suspension in each of the following buffers: BSA buffer (0.1% (w/v) BSA contained in high salt buffer, pH 8.3), high salt buffer (NaCl, 3.7%; KCl, 0.02%; Na2HP04, 0.115%; KH2P04, 0.02%; 0.2% (v/v) Nonidet P-40 [polyoxyethylene (9) p-r-octyl phenol], adjusted to pH 8.3 with 1 M NaOH) and TBS buffer (pH 8.3). Step (3). After the second cycle the beads were resuspended, with vortexing, in 0.5 ml TBST buffer (pH 8.3) containing either goat anti-mouse IgA (a-chain specific) alkaline phosphatase conjugate (Sigma, A-4937) (MM-EIA format [A]) or goat anti-rabbit (whole molecule) alkaline phospbatase conjugate (Sigma, A-8025) (MM-EIA format [B]) at concentrations of l/15,000
colour formation
of MM-EIA formats [A] and [B].
and l/1000 respectively. After 30 min at 23”C, with gentle mixing, the beads were separated magnetically and washed twice as described above. After the second washing cycle the beads were given an additional wash in TBS and then resuspended and equilibrated in 0.5 ml of 12% (v/v) diethanolamine buffer (pH 9.8) for 5 min with gentle mixing. Finally, the beads were separated and resuspended, with vortexing in 250 ~1 of p-nitrophenyl phosphate substrate (diethanolamine buffer containing 0.6 mg PNP substrate (Sigma 104 phosphatase substrate, 104-O ml-‘) and the optical density monitored over time at 405 nm. Elution of bound immunoglobulin from magnetic beads. IgM MAbs were eluted from the beads with
3 M NaI. 10’ beads were mixed, with vortexing, for 10 s with 100 ~1 of 3 M NaI and then incubated for a further 1 min with mixing. The beads were separated magnetically and following aspiration of the supematant, the elution procedure was repeated. The beads were washed three times with PBS and then resuspended in storage buffer at a concentration of 4 x 108 beads ml-‘.
530
C. R. Thornton solani antigens
using both [A] and [B] MM-EIA formats. Increases in mean absorbance values over time were significantly greater than those obtained from control (CPS[ - J) soil extracts (Fig. 2). There were no significant differences in mean absorbance values obtained from (CPS[ - ]) extracts and extract buffer only. Calibration curve
Using the immuno-magnetic bead assay (MM-EIA format [A]) there was a linear relationship between log,0 absorbance at 405 nm and mycelial biomass over the range @40 pg dry mycelium (Fig. 3).
0.5
L.0
I.5
2.0
2.5
Differentiation between anastomosis group isolates and related species by MM-EIA
between R. solani AG isolates and Rhizoctonia spp., by immuno-magnetic bead
Differentiation
Incubation period (h) Fig. 2. Detection of live mycelium of R. soluhi in soil by MM-EIA: absorbance at 405 nm against substrate incubation time. -a- and -m- extracts from soil artificially infested with R. soluni (CPS[ + 1) tested by MM-EIA formats [A] and [B] respectively. -O- and -D- extracts from
uninfested chopped potato soil (CPS[ - 1) tested by MM-EIA formats [A] and [B] respectively. + buffer only. Each point represents the mean of five replicate values (CPS [ + ] and CPS[ - 1) and the mean of three replicate values _+95% confidence interval (extract buffer only). Note that the data from extract buffer alone coincide closely with those from the CPS( - ) treatment.
related
assay, was determined after 30 min incubation in PNP substrate (MM-EIA format [A]). With the exception of the AG2-2 isolate, surface washings from all R. solani anastomosis groups tested positive for mycelial antigens by MM-EIA with the largest absorbance values being achieved with extracts from the group 2-1, 3 and 5 isolates tested (Fig. 4). There was slight recognition of R. tuliparum extracts but no recognition of extracts from the R. carotae and R. cerealis isolates.
RESULTS DISCUSSION
Detection of R. solani antigens in soil
Extracts from soil samples artificially infested with R. sofani (CPS[ + 1) tested positive for detection of R.
Using murine monoclonal antibodies (MAbs) and rabbit (polyclonal) antiserum (PAbs), raised by
20
30
Dry weight mycelium (pg) Fig. 3. MM-EIA (format [A]) calibration curve: loglo absorbance at 405nm, after 2.5 h incubation in PNP substrate, against O-40 pg dry mycelium. Each point represents the mean of three replicate values _+ standard error.
Detection and quantification of Rhizoctoniasolaniin soil
531
MAbs recognise soluble antigens from over twenty separate UK R. cereulis isolates. Recognition of antigens from R. solani AGs 6,8 and 9 has also been (unpub. observations) established. Differentiation of isolates and calibration was not tested using assay format [B]. A soluble immune complex formed between the immunoglobulins and soluble antigen was isolated using magnetic beads coated with antibodies speciftcally targeted against the murine IgM MAbs. The beads, which are available commercially, can be used without prior purification of the MAbs. It was found that beads coated with rat antibodies raised against murine IgM antibodies were most eliicient in isolating the immune complex. The immune complex : was formed within 30 mins, as was binding of the complex to the beads. Concentration of the beads during the isolation step was of paramount importance in abtaining efficient separation, with lower concentrations of Fig. 4. Differentiation of anastomosis groups of A. solani beads (< 10’ ml - ‘) requiring substantially longer and related species by MM-EIA format [A]. Each bar (mean incubations. However, extending the period of the of duplicate values corrected by subtracting mean of binding step resulted in an increase in non-specific absorbance values obtained from buffer only) represents the absorbance at 405 mn obtained after 30 min in PNP binding of soil components that interfered with the substrate. assay. A bead concentration of 10’ ml- I, as recommended by the manufacturers, was found to give maximum complex isolation with minimal Thornton et al. (1993), I have developed novel non-specific binding. While centrifugation of soil extract solutions magnetic microsphere-enzyme immunoassays (MM-EIAs) to detect and quantify Rhizoctonia solani markedly reduced assay interference, non-specific my&al antigens in soil. The assays, developed and binding could not be eliminated by blocking of unoccupied sites on the beads using proteins, e.g. described here, were tested under controlled conditions. Further studies with non-sterile soils will casein and gelatin, prior to the complex isolation determine the performance of the assay under less step. However, non-specific proteins were removed using basic, high salt buffers during the washing artificial conditions. Two assay formats were developed; the first, steps. pH of buffers was extremely important during designated format [A], used a combination of IgM both incubation and washing steps. Low, acidic pH and IgA MAb supematants derived from separate values caused a dissociation of the immune complex murine hybridoma cell lines, while the second [B] from the beads thus all steps were performed at high used a mixture of murine IgM MAb supernatant pH. Several detergents were. tested for use in the and rabbit antiserum. This is the first time, to my washing buffers including Triton X-100 and SDS which were found to be too highly denaturing. knowledge that murine MAbs and rabbit PAbs have been used to develop immuno-magnetic bead assays Nonidet P-40 proved to he the most efficient at to detect and quantify antigens, from a fungal plant reducing non-specific binding as did the addition of BSA, at low concentrations, to the washing buffer. pathogen, in soil. Using format [A] I was able to detect antigens from Interestingly, we found that non-specific binding was lower in samples that used a combination of anastomosis groups (AG) 1,2-l, 3,4, 5 and 7 within 2 h and to construct a calibration curve to quantify murine MAbs and rabbit antiserum. This contrasts sharply with results from micro-tine plate-based my&al biomass of the AG4 isolate R3 to which the double-antibody-sandwich formats using the same antibodies were raised. Sensitivity of the bead assay, compared to other quantitative immuno-assay combination of immunoglobulin species where detection in soil was not possible due to high formats, needs to be tested but is beyond the scope background interference (Thornton et al., 1993). of my paper. However, the major advantage of this Coloured soil extracts did not interfere with the assay is in its speed particularly where extracts from immuno-magnetic bead assay. pure cultures are used. Indeed, it is possible, with this The isolated immune complex was visualised using assay, to rapidly differentiate AG2-2 isolates from secondary antibody-enzyme conjugates. Selection of other AGs (2 h or less). I have found (unpub. the appropriate enzyme system and specificity of the observations), that the assay does not detect antigens detector molecule were extremely important. The from a number of AG2-2 isolates obtained from enzyme horse-radish peroxidase was not found to be different geographical areas world-wide nor do the
532
C. R.
compatible with the bead assay developed here. Alkaline phosphatase conjugates not only resulted in a significant reduction in noise-to-signal ratios but also enabled optical densities to be monitored over time. The assay was amplified with the biotin-extravidin alkaline phosphatase system but little benefit was realised due to increases in background noise. Direct conjugation of enzyme or biotin to the primary antibody(ies) would not only increase the speed of the assay but may also help to reduce interference. The rapidity and sensitivity of the MM-EIA described here was largely determined by the length of antigen extraction. Differentiation between Isolates of R. solani and other Rhizoctonia species grown in vitro was achieved within 2 h with an antigen extraction time of only 5 min being required. As the detection of antigen in soil required longer periods of antigen extractions, 16 h was chosen since this gave maximum assay sensitivity. The water-soluble nature of the antigen meant that lengthy isolation and concentration of antigens from infested soil samples was simplified considerably with the entire procedure being completed in a single unsupervised overnight incubation step. As with any assay, the value of immuno-magnetic bead assay must be judged on the criteria of repeatability and convenience. MM-EIAs, such as the one described here, have considerable advantages over conventional plate trapped antigen enzyme immunoassay systems. Due to the nature of the assay format lengthy coating steps of disposable micro-titre plates are negated and the larger binding surfaces and faster binding kinetics of the beads means that assay processing times are reduced considerably. I have shown that technical challenges such as the elimination of effects of soil, collection and concentration of antigens and sample preparation can be successfully addressed enabling the development of rapid, user-friendly immunoassays for the detection and quantification of soil-borne fungi. The
Thornton assays do not require specialist knowledge and equipment; calorimetric measurement of fungal antigens using an enzyme-chromogen system enables semi-quantitative assessments of biomass to be made provided that adequate controls are included. I have shown that it is possible to quantify the MM-EIA using a simple calibration of fungal biomass. Although the beads are relatively expensive they can be re-used following elution of the bound antibodyantigen complexes using sodium iodide, a gentle and efficient eluting agent (compared with acetic acid). Acknowledgements-I gratefully acknowledge the receipt of an AFRC grant during the course of this study and for
guidance and support of Drs C. A. Gil&an and F. M. Dewey (Universities of Cambridge and Oxford, respectively). Thanks are also due to D. J. Bailey for development of the mycelial disc biomass assay.
REFERENCES Banttari E. E., Clapper D. L., Sheau-Ping Hu, Daws K. M. and Khurana S. M. P. (1991) Rapid magnetic microsphere-enzyme immunoassay for Potato Virus X and Potato Leafroll Virus. Phytopathology, 81, 10391042. Nilsson H., Johansson C. and Scheynius A. (1987) Removal of Langerhans cells from human epidermal cell suspensions by immuno-magnetic particles, Journal of Immunological Methods, 105, 16>169. Pain N. A., Green J. R., Gammie F. and O’Connell R. J. (1994) Immunomaguetic isolation of viable intracellular hyphae of Colletotrichum lindemuthianum (Sacc. & M&n.) Briosi & Cav. from infected bean leaves using a monoclonal antibody. New Phvtoloaist, 127, 223-232. Thornton C. R., Dewey F. M. and &ligan C. A. (1993). Development of monoclonal antibody-based immunological assays for the detection of live propagules of Rhizoctonia solani in soil. Plant Pathology, 42, 763-773. Wipat A., Wellington E. M. H. and Saunders V. A. (1994) Monoclonal antibodies for Streptomyces lividans and their use for immunomagnetic capture of spores from soil. Microbiology, 140, 2067-2076. Wynick D. and Bloom S. R. (1990) Magnetic bead separation of anterior pituitary cells. Neuroendocrinology, 52, 560-565.
Soil Biol. B&hem. Vol. 28, No. 415, pp. 521-532, 19% Copyright 0 1996Ekvicr ScienceLtd Printedin Great Britain. AU rights reserved soo38o717(95)08164-6 0038-0717/% s15.00 + 0.00
DETECTION AND QUANTIFICATION OF RHIZOCTONIA SOLANI IN SOIL BY MONOCLONAL ANTIBODY-BASED IMMUNO-MAGNETIC BEAD ASSAY C. R. THORNTON Department Plant Sciences, University of Cambridge, Downing St., Cambridge CB2 3EA, U.K. (Accepted 20 November 1995)
Sammy-Murine monoclonal antibodies (MAbs) of the immunoglobulin classes IgM and IgA, from two different hybridoma cell lines, and rabbit (polyclonal) antisermn raised against mycehal antigens from an AG4 isolate of R. sofoni were used to develop magnetic microsphere-enzyme immunoassays (MM-EIAs) for the detection and quantitication of the pathogen in soil. The assay can also be used to differentiate R. solani anastomosis group (AG) 2-2 isolates from other AG isolates grown
in vitro. My&ii
antigens were solubilised in saline buffer containing detergent and following high speed centrifugation soluble antigen was delineated by the addition either of a mixture of specificmurine MAbs
of diffemnt immunoglobulin classes or a mixture of murine MA\, supematant and rabbit antiserum. Due to the low concentration of reactants a soluble immune complex was formed. The soluble complex was isolated by the addition of magnetic beads coated with antibodies that specitically recognised the murine IgM MAbs. The bound antibody-antigen complex was then detected using a commercial antibody-enzyme conjugate and chromogen. Copyright 0 1996 Elsevier Science Ltd
INTRODUCTION
Thornton et al. (1993) reported the development of monoclonal antibodies (MAbs) and MAb-based immunological assays for the detection of Rhizoctonia solani in soil. These assays involve the immobilisation of mycelial antigens onto solid supports (PVC micro-titre wells or PVDF membrane) using an overnight incubation step and subsequent visualisation of bound antigen by enzyme-linked immunosorbent assay (ELISA). My aim is to communicate the development of a novel immunomagnetic bead assay for the detection and quantification of R. sofani mycelial antigens in soil. Magnetic beads have been employed successfully in the medical field but have, to date, been used exclusively as a means of selective cell separation (e.g. Nilsson et al., 1987; Wynick and Bloom, 1990). Wipat et al. (1994) reported the use of MAb-coated beads in the immunomagnetic capture and concentration of spores of the bacterium Streptomyces liuidans in soil while Pain et al. (1994) used immunomagnetic separation to isolate viable intracellular hyphae of Colletotrichum linakmuthianum from infected bean leaves using MAbs. The incorporation of magnet&d beads into magnetic microsphere-enzyme immunoassay (MM-EIA) formats has been associated most notably with the detection and quantification of plant viruses (e.g. Banttari et al., 1991). To my knowledge, no studies
have reported the detection of fungal antigens in soil using an immune-magnetic bead assay. One of the most significant rate determining steps in the development of sensitive immunoassays for the detection of soil-borne fungi is the extraction of their antigens. Most assays require lengthy extraction and concentration procedures often involving nutrient enrichment, sieving, flotation, centrifugation and filtration. The immunoassays themselves often require lengthy processing times. Since most enzyme immunoassays use micro-titre plates or membranes as the immunosorbent surface, they sulfer from slow binding kinetics and small surface areas. In contrast, enzyme immunoassays that incorporate magnetic particles (l-10 pm) have larger binding surfaces and faster binding kinetics. Antibody-coated magnetic beads have the added advantage of utilising a separation step that is uniquely suited to efficiently extracting antigens from samples (l-2 ml) that contain particulate debris. Thus, it is possible to develop immunoassays that combine the extraction, concentration and detection of antigens into a single relatively simple and rapid step. A magnetic field can then be applied that pulls the beads and bound antigen to the side of a micro-centrifuge tube and the remaining soluble and particulate debris can then be decanted or aspirated. The bound antibody-antigen complex is subsequently quantified by the addition of an antibody-enzyme conjugate and chromogen using 521
528
C. R. Thornton
procedures similar to those employed double-antibody-sandwich ELISAs.
by other
METHODS
Fungal culture
Isolates of Rhizoctonia solani anastomosis groups (Table 1) were maintained on potato dextrose agar (PDA, Oxoid CM139) either in Petri-dishes or on slants at 23°C. Development of monoclonal antibodies
The R. solani-specific murine monoclonal antibodies (MAbs) and rabbit (polyclonal) antiserum, used were developed by Thornton et al. (1993); murine hybridoma cell lines EEl and EH2 that produce immunoglobulins of the classes IgM and IgA respectively, and rabbit antiserum, were raised against mycelial antigens from an R. solani AG4 isolate R3. Detection and quanttjkation of R. solani antigens in artificially infested soil by MAb-based immuno-magnetic bead assay Preparation of arttficially infested chopped potato soil. Three-hundred g sieved (0.5-l mm) and
air-dried loam soil (Cambridge University Field Station), and 75 g finely chopped potato (wet weight) were mixed thoroughly with 45 ml reverse osmosis water (R.0.H20) and autoclaved in 250 ml Erlenmeyer flasks for two consecutive 1 h periods at 121°C. Flasks were inoculated with six 3 cm mycelial plugs taken from the leading edge of 5-d-old PDA Petri-dish cultures of either R. solani (CPS( + )) or were inoculated with a similar number of uncolonised plugs (CPS( - )). The flasks were kept in the dark, at 23°C for 2 weeks. Extraction of antigen and preparation of standard curve. Five replicate 5 g fresh samples from
artificially infested and control flask cultures were placed in separate 20 ml polyvials containing 5 ml of PBST buffer (PBS, NaCl, 0.8%; KCl, 0.02%; Na2HP04, 0.115%; KH2P04, 0.02%; PBST, PBS + 0.05% (v/v) Tween-20 [polyoxyethylene(20)sorbitan monolaurate]) adjusted to Table I. Details of fungal cultures Organism Rhizockmia camtoe Rhizockmia cerealis Rhizoctonia solani
AG
I 2-l 2-2 3 4 4 5 7
R
tuliparum
Isolate
numtw
168256 RI 303 154/R20 R5 303 156/R2 1 303 138/R22 303162/R23 R3 303 159/R24 303161/R25 236841
RB, R. Baker; FMD, F. M. Dewey; IMI, International Institute, Egham, Surrey.
Source IMI FMD IMI RB IMI IMI IMI RB IMI IMI IMI Mycological
pH 8.3 with 1 M NaOH and the vials incubated for 16 h, at 23”C, with mixing. The contents of the vials were centrifuged, at 12,000 g for 20 min, and 875 ~1 of extract transferred to 1.5 ml micro-centrifuge tubes for immediate assay by magnetic microsphereenzyme immunoassay (MM-EIA). A standard curve, relating biomass of mycelium (pg dry mycelium) to absorbance value, was constructed to determine the sensitivity of the enzyme immunoassay. Sterile nitro-cellulose millipore filter papers (dia 0.44 cm, pore size 0.45 pm), overlaying PDA in 9 cm Petri-dish plates, were inoculated centrally with single 3 cm dia plugs taken from the leading edge of 5-d-old PDA cultures of R. solani. After approximately 5 days at 23°C the filters were extracted with forceps and discs of mycelium punched from the filters using a 501 micro-pipette (Camlab, Cambridge, U.K.). The mycelial discs were separated from the filters and were placed in 1 ml volumes of PBST (pH 8.3) contained in micro-centrifuge tubes and homogenised for approximately 5 min using a hand-held Eppendorf homogeniser. Cellular debris was removed by centrifugation at 12,000 g for 20 min and the supematant, containing soluble antigen, transferred to clean micro-centrifuge tubes for immediate assay by MM-EIA (format [A]) described below. Recognition of anastomosis group isolates. Differentiation of AG isolates of R. solani and related Rhizoctonia spp., by immuno-magnetic bead assay, was tested using cell-free PBST (pH 8.3) surface washings of antigens from slant cultures, diluted IO-fold into PBST, according to the MM-EIA format [A] described below. 1Uh4-EIA formats. Aliquots (25 ~1) of magnetic beads (Dynabeads M-450 rat anti-mouse IgM [Dynal (UK)] Ltd., No. 110.15) coated with rat monoclonal antibodies that specifically recognise the p heavy chain of mouse IgM immunoglobulins (approximately 10’ beads ml - ’storage buffer (0.1 M NaP04 buffer, pH7.4, containing 0.1% (w/v) human serum albumin and 0.02% (v/v) NaNj) were placed in 1.5 ml micro-centrifuge tubes. The beads were washed four times, by vortexing, with 1 ml volumes of TBS buffer (NaCl, 0.8%; KCI, 0.02%; Tris, 0.3%; pH 8.3). After each washing step the beads were seperated using a magnetic particle concentrator (Dynal, MPC-M) and the washing supematant removed by aspiration. Washed beads were finally resuspended, with vortexing, in TBST buffer (TBS + 0.05% (v/v) Tween-20, pH 8.3) with a volume equivalent to the starting volume of storage buffer. Two MM-EIA formats were tested (Fig. 1); the first, designated format [A] used a mixture of murine IgM and IgA MAbs while the second (format [B]) used a mixture of murine IgM MAbs and rabbit antiserum as follows: Step (1). To each of the tubes containing 875 ~1 of antigen extract were added either 50 ~1 each of EEl (IgM) and EH2 (IgA) MAb supematants (MM-EIA
Detection and quantiiication of Rhizoctonia solani in soil
FORMAT
529
FORMAT
[A]
colour forktion Fig. 1. Diagramatic representation
format [A]) or 99 ~1 of EEl MAb supernatant and 1 ~1 of rabbit antiserum (MM-EIA format [B]). Solutions were mixed thoroughly, by vortexing, and incubated with gentle mixing for 30 min at 23°C. Step (2). Washed beads (25 ~1) were added to each tube and after thorough vortexing the mixtures were kept for 30 min at 23°C with gentle agitation. After magnetic separation, the beads were washed free of unbound protein (1 ml each wash) by two cycles of separation and re-suspension in each of the following buffers: BSA buffer (0.1% (w/v) BSA contained in high salt buffer, pH 8.3), high salt buffer (NaCl, 3.7%; KCl, 0.02%; Na2HP04, 0.115%; KH2P04, 0.02%; 0.2% (v/v) Nonidet P-40 [polyoxyethylene (9) p-r-octyl phenol], adjusted to pH 8.3 with 1 M NaOH) and TBS buffer (pH 8.3). Step (3). After the second cycle the beads were resuspended, with vortexing, in 0.5 ml TBST buffer (pH 8.3) containing either goat anti-mouse IgA (a-chain specific) alkaline phosphatase conjugate (Sigma, A-4937) (MM-EIA format [A]) or goat anti-rabbit (whole molecule) alkaline phospbatase conjugate (Sigma, A-8025) (MM-EIA format [B]) at concentrations of l/15,000
colour formation
of MM-EIA formats [A] and [B].
and l/1000 respectively. After 30 min at 23”C, with gentle mixing, the beads were separated magnetically and washed twice as described above. After the second washing cycle the beads were given an additional wash in TBS and then resuspended and equilibrated in 0.5 ml of 12% (v/v) diethanolamine buffer (pH 9.8) for 5 min with gentle mixing. Finally, the beads were separated and resuspended, with vortexing in 250 ~1 of p-nitrophenyl phosphate substrate (diethanolamine buffer containing 0.6 mg PNP substrate (Sigma 104 phosphatase substrate, 104-O ml-‘) and the optical density monitored over time at 405 nm. Elution of bound immunoglobulin from magnetic beads. IgM MAbs were eluted from the beads with
3 M NaI. 10’ beads were mixed, with vortexing, for 10 s with 100 ~1 of 3 M NaI and then incubated for a further 1 min with mixing. The beads were separated magnetically and following aspiration of the supematant, the elution procedure was repeated. The beads were washed three times with PBS and then resuspended in storage buffer at a concentration of 4 x 108 beads ml-‘.
530
C. R. Thornton solani antigens
using both [A] and [B] MM-EIA formats. Increases in mean absorbance values over time were significantly greater than those obtained from control (CPS[ - J) soil extracts (Fig. 2). There were no significant differences in mean absorbance values obtained from (CPS[ - ]) extracts and extract buffer only. Calibration curve
Using the immuno-magnetic bead assay (MM-EIA format [A]) there was a linear relationship between log,0 absorbance at 405 nm and mycelial biomass over the range @40 pg dry mycelium (Fig. 3).
0.5
L.0
I.5
2.0
2.5
Differentiation between anastomosis group isolates and related species by MM-EIA
between R. solani AG isolates and Rhizoctonia spp., by immuno-magnetic bead
Differentiation
Incubation period (h) Fig. 2. Detection of live mycelium of R. soluhi in soil by MM-EIA: absorbance at 405 nm against substrate incubation time. -a- and -m- extracts from soil artificially infested with R. soluni (CPS[ + 1) tested by MM-EIA formats [A] and [B] respectively. -O- and -D- extracts from
uninfested chopped potato soil (CPS[ - 1) tested by MM-EIA formats [A] and [B] respectively. + buffer only. Each point represents the mean of five replicate values (CPS [ + ] and CPS[ - 1) and the mean of three replicate values _+95% confidence interval (extract buffer only). Note that the data from extract buffer alone coincide closely with those from the CPS( - ) treatment.
related
assay, was determined after 30 min incubation in PNP substrate (MM-EIA format [A]). With the exception of the AG2-2 isolate, surface washings from all R. solani anastomosis groups tested positive for mycelial antigens by MM-EIA with the largest absorbance values being achieved with extracts from the group 2-1, 3 and 5 isolates tested (Fig. 4). There was slight recognition of R. tuliparum extracts but no recognition of extracts from the R. carotae and R. cerealis isolates.
RESULTS DISCUSSION
Detection of R. solani antigens in soil
Extracts from soil samples artificially infested with R. sofani (CPS[ + 1) tested positive for detection of R.
Using murine monoclonal antibodies (MAbs) and rabbit (polyclonal) antiserum (PAbs), raised by
20
30
Dry weight mycelium (pg) Fig. 3. MM-EIA (format [A]) calibration curve: loglo absorbance at 405nm, after 2.5 h incubation in PNP substrate, against O-40 pg dry mycelium. Each point represents the mean of three replicate values _+ standard error.
Detection and quantification of Rhizoctoniasolaniin soil
531
MAbs recognise soluble antigens from over twenty separate UK R. cereulis isolates. Recognition of antigens from R. solani AGs 6,8 and 9 has also been (unpub. observations) established. Differentiation of isolates and calibration was not tested using assay format [B]. A soluble immune complex formed between the immunoglobulins and soluble antigen was isolated using magnetic beads coated with antibodies speciftcally targeted against the murine IgM MAbs. The beads, which are available commercially, can be used without prior purification of the MAbs. It was found that beads coated with rat antibodies raised against murine IgM antibodies were most eliicient in isolating the immune complex. The immune complex : was formed within 30 mins, as was binding of the complex to the beads. Concentration of the beads during the isolation step was of paramount importance in abtaining efficient separation, with lower concentrations of Fig. 4. Differentiation of anastomosis groups of A. solani beads (< 10’ ml - ‘) requiring substantially longer and related species by MM-EIA format [A]. Each bar (mean incubations. However, extending the period of the of duplicate values corrected by subtracting mean of binding step resulted in an increase in non-specific absorbance values obtained from buffer only) represents the absorbance at 405 mn obtained after 30 min in PNP binding of soil components that interfered with the substrate. assay. A bead concentration of 10’ ml- I, as recommended by the manufacturers, was found to give maximum complex isolation with minimal Thornton et al. (1993), I have developed novel non-specific binding. While centrifugation of soil extract solutions magnetic microsphere-enzyme immunoassays (MM-EIAs) to detect and quantify Rhizoctonia solani markedly reduced assay interference, non-specific my&al antigens in soil. The assays, developed and binding could not be eliminated by blocking of unoccupied sites on the beads using proteins, e.g. described here, were tested under controlled conditions. Further studies with non-sterile soils will casein and gelatin, prior to the complex isolation determine the performance of the assay under less step. However, non-specific proteins were removed using basic, high salt buffers during the washing artificial conditions. Two assay formats were developed; the first, steps. pH of buffers was extremely important during designated format [A], used a combination of IgM both incubation and washing steps. Low, acidic pH and IgA MAb supematants derived from separate values caused a dissociation of the immune complex murine hybridoma cell lines, while the second [B] from the beads thus all steps were performed at high used a mixture of murine IgM MAb supernatant pH. Several detergents were. tested for use in the and rabbit antiserum. This is the first time, to my washing buffers including Triton X-100 and SDS which were found to be too highly denaturing. knowledge that murine MAbs and rabbit PAbs have been used to develop immuno-magnetic bead assays Nonidet P-40 proved to he the most efficient at to detect and quantify antigens, from a fungal plant reducing non-specific binding as did the addition of BSA, at low concentrations, to the washing buffer. pathogen, in soil. Using format [A] I was able to detect antigens from Interestingly, we found that non-specific binding was lower in samples that used a combination of anastomosis groups (AG) 1,2-l, 3,4, 5 and 7 within 2 h and to construct a calibration curve to quantify murine MAbs and rabbit antiserum. This contrasts sharply with results from micro-tine plate-based my&al biomass of the AG4 isolate R3 to which the double-antibody-sandwich formats using the same antibodies were raised. Sensitivity of the bead assay, compared to other quantitative immuno-assay combination of immunoglobulin species where detection in soil was not possible due to high formats, needs to be tested but is beyond the scope background interference (Thornton et al., 1993). of my paper. However, the major advantage of this Coloured soil extracts did not interfere with the assay is in its speed particularly where extracts from immuno-magnetic bead assay. pure cultures are used. Indeed, it is possible, with this The isolated immune complex was visualised using assay, to rapidly differentiate AG2-2 isolates from secondary antibody-enzyme conjugates. Selection of other AGs (2 h or less). I have found (unpub. the appropriate enzyme system and specificity of the observations), that the assay does not detect antigens detector molecule were extremely important. The from a number of AG2-2 isolates obtained from enzyme horse-radish peroxidase was not found to be different geographical areas world-wide nor do the
532
C. R.
compatible with the bead assay developed here. Alkaline phosphatase conjugates not only resulted in a significant reduction in noise-to-signal ratios but also enabled optical densities to be monitored over time. The assay was amplified with the biotin-extravidin alkaline phosphatase system but little benefit was realised due to increases in background noise. Direct conjugation of enzyme or biotin to the primary antibody(ies) would not only increase the speed of the assay but may also help to reduce interference. The rapidity and sensitivity of the MM-EIA described here was largely determined by the length of antigen extraction. Differentiation between Isolates of R. solani and other Rhizoctonia species grown in vitro was achieved within 2 h with an antigen extraction time of only 5 min being required. As the detection of antigen in soil required longer periods of antigen extractions, 16 h was chosen since this gave maximum assay sensitivity. The water-soluble nature of the antigen meant that lengthy isolation and concentration of antigens from infested soil samples was simplified considerably with the entire procedure being completed in a single unsupervised overnight incubation step. As with any assay, the value of immuno-magnetic bead assay must be judged on the criteria of repeatability and convenience. MM-EIAs, such as the one described here, have considerable advantages over conventional plate trapped antigen enzyme immunoassay systems. Due to the nature of the assay format lengthy coating steps of disposable micro-titre plates are negated and the larger binding surfaces and faster binding kinetics of the beads means that assay processing times are reduced considerably. I have shown that technical challenges such as the elimination of effects of soil, collection and concentration of antigens and sample preparation can be successfully addressed enabling the development of rapid, user-friendly immunoassays for the detection and quantification of soil-borne fungi. The
Thornton assays do not require specialist knowledge and equipment; calorimetric measurement of fungal antigens using an enzyme-chromogen system enables semi-quantitative assessments of biomass to be made provided that adequate controls are included. I have shown that it is possible to quantify the MM-EIA using a simple calibration of fungal biomass. Although the beads are relatively expensive they can be re-used following elution of the bound antibodyantigen complexes using sodium iodide, a gentle and efficient eluting agent (compared with acetic acid). Acknowledgements-I gratefully acknowledge the receipt of an AFRC grant during the course of this study and for
guidance and support of Drs C. A. Gil&an and F. M. Dewey (Universities of Cambridge and Oxford, respectively). Thanks are also due to D. J. Bailey for development of the mycelial disc biomass assay.
REFERENCES Banttari E. E., Clapper D. L., Sheau-Ping Hu, Daws K. M. and Khurana S. M. P. (1991) Rapid magnetic microsphere-enzyme immunoassay for Potato Virus X and Potato Leafroll Virus. Phytopathology, 81, 10391042. Nilsson H., Johansson C. and Scheynius A. (1987) Removal of Langerhans cells from human epidermal cell suspensions by immuno-magnetic particles, Journal of Immunological Methods, 105, 16>169. Pain N. A., Green J. R., Gammie F. and O’Connell R. J. (1994) Immunomaguetic isolation of viable intracellular hyphae of Colletotrichum lindemuthianum (Sacc. & M&n.) Briosi & Cav. from infected bean leaves using a monoclonal antibody. New Phvtoloaist, 127, 223-232. Thornton C. R., Dewey F. M. and &ligan C. A. (1993). Development of monoclonal antibody-based immunological assays for the detection of live propagules of Rhizoctonia solani in soil. Plant Pathology, 42, 763-773. Wipat A., Wellington E. M. H. and Saunders V. A. (1994) Monoclonal antibodies for Streptomyces lividans and their use for immunomagnetic capture of spores from soil. Microbiology, 140, 2067-2076. Wynick D. and Bloom S. R. (1990) Magnetic bead separation of anterior pituitary cells. Neuroendocrinology, 52, 560-565.